Utility of a modified vascular corrosion casting technique in the diagnosis of fetal total anomalous pulmonary venous connection

Total anomalous pulmonary venous connection (TAPVC) is a rare congenital cardiac malformation, and prenatal detection of TAPVC malformation remains a challenging. TAPVC can be easily missed or misdiagnosed in prenatal examinations. This study was aimed to use the modified vascular corrosion casting technique to prepare fetal cardiovascular casts with TAPVC and investigate the utility of cardiovascular casting for the demonstration of fetal TAPVC. The retrospective study enrolled twenty fetuses (22 to 29 + 4 gestational weeks) with TAPVC diagnosed by prenatal echocardiography and casting technique from May 2015 to May 2020. Pre- and postnatal medical records, including results obtained by prenatal ultrasound, postpartum computed tomography angiography, as well as anatomic and cardiovascular casting findings were carefully reviewed and analyzed. In twenty cases, 80% (16/20) had intra- or extracardiac malformations. The TAPVC types were supracardiac (n = 8), cardiac (n = 6), infracardiac (n = 4), and mixed (n = 2). The diagnosis of 1 case each of supracardiac and cardiac TAPVC was modified to partial anomalous pulmonary venous connection; additionally, 4 malformations were missed and 2 were misdiagnosed, including an anomalous left brachiocephalic vein in supracardiac TAPVC, abnormal inflow of the hepatic vein and a double inferior vena cava in infracardiac TAPVC; and bilateral ductus arteriosus in infracardiac TAPVC; a tetralogy of Fallot in cardiac TAPVC that was corrected to right ventricular double outlet; and an absence of ductus arteriosus that was misdiagnosed as slim ductus arteriosus. Comparing with ultrasound, casting technique has its own superiority in exhibiting TAPVC abnormalities, especially in certain types such as course, origin and absence abnormalities of ductus. Postpartum cardiovascular casts can accurately depict the branch structure of the heart’s larger vessels, and may be used as a clinical assessment and teaching method in complex cardiac malformations.

www.nature.com/scientificreports/ remains clinically challenging for obstetricians; in a retrospective analysis of birth records from 1998 to 2004, only 1.9% of TAPVC cases (8 in 424) were prenatally diagnosed 13 . Because of its noninvasiveness and convenience and real-time monitoring capabilities, prenatal ultrasound is becoming the preferred method for diagnosing TAPVC, especially with the development of new scanning technologies such as color flow Doppler imaging and spatiotemporal image correlation, which has improved the diagnosis rate. However, given the lack of awareness of this rare malformation and the fact that it is often accompanied by complex cardiac and intracardiac malformations, TAPVC can be easily overlooked or misdiagnosed 14 .
In the present study, we retrospectively analyzed the clinical data of 20 cases of fetal TAPVC diagnosed by prenatal ultrasound, and compared the results of antenatal echocardiography with postpartum echocardiography, postpartum computed tomography angiography (CTA), and anatomic findings to establish the characteristics of different types of TAPVC and their venous return. In order to clearly display the 3D structure of fetal vessels, modified cardiovascular casts of some specimens were also used for postpartum diagnosis.

Materials and methods
Study participants. The study was approved by the ethics committee of Xiangyang No. 1 People's Hospital Affiliated with Hubei University of Medicine. All pregnant women enrolled in our study provided written, informed consent. We retrospectively analyzed 20 cases of TAPVC diagnosed by ultrasound before delivery between May 2015 and May 2020. The gestational age ranged from 22 + 4 to 29 + 6 weeks (mean, 25 + 3 weeks), and maternal age was between 19 and 36 years (mean, 27.6 years).
Prenatal ultrasound. Prenatal ultrasound was performed according to guidelines of the International Society of Ultrasound in Obstetrics and Gynecology and American Society of Echocardiography using a Voluson E8 or 730 ultrasound system (GE Medical Systems, Zipf, Austria) equipped with RM6C, C4-8, and C4-8D transducers (4)(5)(6)(7)(8). The number, location, and specific PV drainage routes were visualized in a short axis section of the heart. Postnatal diagnosis. Postpartum diagnosis was based on ultrasound, CTA, surgery, anatomic examination, and cardiovascular casts. For pregnant women who chose natural delivery, postpartum diagnosis was made according to the results of the operation or postpartum ultrasound. Some pregnant women chose to have labor induced because of complex intra-or extracardiac malformations in the fetus, and donated the specimens to our maternal and fetal medical center. In these cases, the postpartum diagnosis was based on CTA, anatomy, and cardiovascular casts. Postpartum ultrasound was performed using a GE Vivid 7 ultrasound system (GE Medical Systems, Zipf, Austria) with a probe frequency of 2.5-12 MHz. Reflux into the left atrium and cardiac malformations were visualized by analyzing Cardiac segments and aortic cross sections of the superior sternal fossa.
Modified cardiovascular casting. For cardiovascular casting, the specific steps are as follow: (1) Fill in the fetal basic information record form and documentary; (2) The fetal weight, body length, head circumference, chest circumference, abdominal circumference, eye distance, upper limb length and lower limb length were measured and recorded; (3) Take photos before casting; (4) Incision of abdominal wall, separation of umbilical vein, umbilical vein intubation; (5) The left umbilical artery was cut off; (6) Heparin (5-10 ml) and acetone (20-50 ml) were injected through umbilical vein catheterization to wash blood and blood clots in cardiovascular system. The injection amount of acetone was adjusted according to the situation of blood washing until no blood flowed out. The mixture of self-coagulating denture powder and denture water was perfused slowly with pressure of 30 ~ 60 ml, and the injection time was controlled at about 30 min. Finally, the mixture of self-setting denture powder and denture water was infused with a pressure syringe for about 10 h; (7) After 24 h, the fetal specimens perfused with the mixed reagent of self-setting denture powder and denture water were immersed in 30% hydrochloric acid solution; (8) After about 2 weeks, the perfused fetal specimens were taken out from hydrochloric acid solution, and the corroded soft tissues were washed carefully with running water. The soft tissues that were not washed clean were washed carefully with syringe until there was no soft tissue on the surface of cardiovascular cast; (9) The fetal cardiovascular cast specimens were registered and photographed. The ratio of materials used for perfusion was as follows:

Results
General and clinical characteristics of cases. A total of 20 fetuses with a prenatal diagnosis of TAPVC were included in the analysis. The median age of the pregnant women was 27.6 years (range, 19-36 years), and the median gestational age at diagnosis was 25 weeks (range, 22-29 weeks). Of the 20 cases, 18 were confirmed as TAPVC by postpartum diagnosis, and the other 2 (cases 4 and 15) were modified to partial anomalous pulmonary venous connection. The different types of TAPVC were supracardiac, 44% (8/18); cardiac, 22% (4/18); infracardiac, 22% (4/18); and mixed, 11% (2/18). Intra-or extracardiac malformations were observed in 78%  Fig. 1), which was confirmed by postpartum findings (Figs. 2, 3, 4). In the 2 cases of cardiac TAPVC, right atrial isomerism, diaphragmatic hernia, a single atrium, pulmonary atresia, abnormal inflow and atrial isomerism syndrome, levoversion, atrial septal defect, functional single ventricle, double-outlet single ventricle, and double-inlet single ventricle were prenatally diagnosed (case 8, Fig. 5; case 17, Fig. 6 A, B) The apex of the heart was to the right, there was a single ventricle, the gallbladder and stomach were in the right and left abdomens, respectively, and a spleen was lacking. GB gallbladder, L-LU left lung, R-LU right lung, ST stomach.

Discussion
TAPVC can be accurately diagnosed after birth by combining 2-dimensional echocardiography with color Doppler imaging. However, prenatal diagnosis remains a clinical challenge, and there have few studies addressing this issue 11,15,16 . In the early stage of pregnancy, fetal blood mostly circulates through the ductus arteriosus and only a small portion of pulmonary blood flows into the left atrium via the PV; as no hemodynamic changes are observed at this time, diagnosing TAPVC is difficult. During the middle and late periods of pregnancy, pulmonary blood flow into the right atrium via the PV is increased, including in the fetus with TAPVC. At this point, pulmonary hypertension can easily occur and can lead to right heart failure postnatally and death. Therefore, prenatal diagnosis of fetal TAPVC is extremely important 13,17,18 .
The results of this retrospective study carried out at our Maternal-Fetal Medical Center showed that TAPVC can be identified by targeted fetal echocardiography, which can provide important 2D and spectral information that can be used for diagnosis. Moreover, we demonstrated that a modified cardiovascular cast can be used to display the 3D structure of the PV, which can also guide prenatal diagnosis. Among the 20 fetuses with a prenatal diagnosis of TAPVC, 18 were confirmed by postpartum diagnosis, and 2 cases were modified to PAPVC. Both of these showed complex intracardiac anatomy, with right atrial isomerism, endocardial cushion defect, a single ventricle, interrupted aortic arch, and PAPVC with the right PV connected to the right superior vena cava observed in 1 case and right atrial isomerism, endocardial cushion defect, functional single ventricle, double-outlet single ventricle, pulmonary artery stenosis, double superior vena cava, and PAPVC with the right PV connected to the right atrium observed in the other. Besides the 4 cases of isolated TAPVC, 14/18 cases (78%) were complicated by intra-or extracardiac malformations, and 61% (11/18) were complicated by heterotaxia syndrome; as the latter can lead to atrial heterogeneity and a change in the position of the atrium, it is critical for clinicians to identify the true left and right atria, which may be a key step in the diagnosis of TAPVC. Following pre-and postnatal diagnoses, fetal echocardiography showed no direct connection between the PV and left atrium but revealed the convergence of PVs at the back of the atrium to form a common vein stem; an increased distance between the posterior wall of the atrium and descending aorta; and a visible ascending or descending vertical vein. These features not only exist in a fetus with complex intra-or extracardiac malformations, but also in isolated TAPVC.
Our modified cardiovascular cast is another method for postmortem evaluation of fetal TAPVC. In China, our team was the first to improve and patent the modified vascular casting technology. We previously demonstrated that the cardiovascular cast can accurately show various complex fetal cardiac malformations, which can facilitate prenatal diagnosis and reduce the risk of overlooking or misdiagnosing the Complex cardiac malformations 19,20 .
Several studies have evaluated the clinical utility of postmortem magnetic resonance imaging (MRI) and micro-CT [21][22][23] . www.nature.com/scientificreports/ However, casting may better reveal some rare malformations, especially smaller branching anomalies that are difficult to detect by other imaging modalities. In the future, this new approach of casting combined with micro-CT or MRI may be used to develop 3D digital models for clinical assessment and teaching.

Conclusion
The results of our study demonstrate that prenatal ultrasound can be used to accurately diagnose most cases of fetal TAPVC. By analyzing the echocardiography findings, the specific type of fetal TAPVC can be identified. Identifying the PV drainage route is critical for diagnosing different types of TAPVC. Finally, a postpartum cardiovascular cast can accurately depict the branch structure of the heart's major vessels, which can guide prenatal diagnosis of TAPVC for timely management that can lead to an improved outcome.

Data availability
The data used to support the findings of this study are included within the article.
Received: 28 October 2020; Accepted: 4 May 2021 Table 3. Comparing the modified corrosion technique to the currently available fetal imaging methods on TAPVC.

Modified corrosion technique
Cardiovascular casting technique has its own superiority in exhibiting TAPVC abnormalities, especially in certain types such as course, origin and absence abnormalities of ductus All cases of pulmonary veins are perfectly delineated Autopsy For complex congenital heart malformations, the display of vessels course is not as good as that of cardiovascular casting 8

Prenatal ultrasound
Prenatal ultrasound is limited by maternal amniotic fluid content, fetal position and ultrasound imaging itself, which is easy to cause missed diagnosis and misdiagnosis of TAPVC The diagnosis of 1 case each of supracardiac and cardiac TAPVC was modified to partial anomalous pulmonary venous connection; additionally, 4 malformations were missed and 2 were misdiagnosed Postnatal echocardiography It can't display the complex vascular malformation stereoscopically and completely 12 Postpartum computed tomography angiography The imaging results is closely related to the effect of cadaveric radiography after induced labor, which is prone to the reconstruction artifacts caused by the overflow of contrast medium 5